Patent UA-104525-C2 (UA)
Title: Method for early diagnostics of fibrosis of liver, kidney, heart at experimental hyperthyroidism and hypothyriodism
IPC: G01N33/53
Filed: 2012-11-16
Abstract: The invention relates to medicine, in particulµar to endocrinology, and can be used for early diagnostics of development of fibrosis of tissue at hyperthyroidism and hypothyroidism. In the method according to the invention states of hyperthyroidism and hypothyroidism are modeled in rats, with sampling peripheral blood, with centrifugation at 1,000 rpmfor 5 min with removal of supernatant fluid, with washing blood clot with phosphate buffer, centrifugation is repeated at 1000 rev/min for 5 min, cellular sediment is re-suspended with solution of pre-extraction buffer and with inhibitors of proteases, with incubation on ice for 10 min. Content of test tubes is carefully mixed, with centrifugation once again at 12,000 rpm for 1 min, cytoplasm supernatant is removed with addition of dithiothreitol solution, proteases inhibitor and extraction buffer to the deposit, obtained solution is incubated in ice for 15 min, with mixing it each 5 minutes, with centrifugation at 14,000 rpm at 4 °C for 10 min, supernatant is separated and moved to new test tubes, with measurement of concentration of proteins in nuclear extraction obtained at wavelength 560 nm, with comparison of data to control. Bull serum albumin is used, with different concentrations (0 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1.0 mg/ml, 1.4 mg/ml, 2 mg/ml). Then enzymatic activity of DNA-methyltransferase (DNMT) is determined, that for to the wells samples are placed, those are added in volume according to concentration of protein, to the wells with positive control solution of positive control with buffer is added, to the wells with negative control - inhibitor DNMT procainamide chloride at different concentrations, 10 µg/ml, 1 µg/ml, and to one well as control only buffer is added, after that content of the wells is mixed, with covering with parafilm and incubation for 90 min at 37 °C, with removal of content of the wells and with washing three times with washing buffer, with adding 50 µl of antibodies to each well for holding DNMT enzyme with incubation for 60 min at room temperature with shaking, with removal of content of the wells and washing those 3-4 times with washing buffer, again 50 µl of antibodies is added to each well for reveal of enzyme, with incubation at room temperature for 30 min, with removal of content of the wells and washing those 4-5 times with washing buffer, with adding 100 µl of developing solution to each well, incubation in dark till blue color for 5 min, with adding 50 µl of stop-reagent to each well, with measurement of optical density at wavelength 450 nm. Then activity of DNMT in samples is calculated, and when its values are above norm and concentration of thyroid hormones is changed the presence of fibrous re-modulation of tissues is stated, and when value of DNMT activity is lower or equal to norm and concentration of hormones of thyroid gland is lower and above the norm, the absence of fibrous changes or non-malignant character of course of hyperthyroidism or hypothyroidism is stated.